Field of the Invention
The invention relates to a variant polypeptide having chymosin activity. The invention also relates to a nucleic acid sequence encoding such a variant, to a recombinant expression vector a said nucleic acid construct and to a recombinant host cell comprising a said expression vector. Further, the invention relates to a method for producing a chymosin via use of such a host cell. Also, the invention relates to a method of producing a chymosin polypeptide variant. The invention further relates to a composition comprising a chymosin variant, to use of such a chymosin variant or chymosin variant-containing composition in the preparation of a cheese, to a process for the production of a cheese and to the resulting cheese.
Description of Related Art
Enzymatic coagulation of milk by milk clotting enzymes, such as chymosin and pepsin, is one of the most important processes in the manufacture of cheeses. Enzymatic milk coagulation is a two-phase process: a first phase where a proteolytic enzyme, chymosin or pepsin, attacks kappa-casein, resulting in a metastable state of the casein micelle structure and a second phase, where the milk subsequently coagulates and forms a coagulum. Chymosin (group 3.4.23.4 according to the Enzyme Nomenclature, 1992 of the International Union of Biochemistry and Molecular Biology, IUBMB) and pepsin (EC 3.4.23.1), the milk clotting enzymes of the mammalian stomach, are aspartic proteases belonging to a broad class of peptidases. Aspartic proteases are found in eukaryotes, retroviruses and some plant viruses.
When produced in the gastric mucosal cells, chymosin and pepsin occur as enzymatically inactive pre-prochymosin and pre-pepsinogen, respectively. When chymosin is excreted, an N-terminal peptide fragment, the pre-fragment (signal peptide) is cleaved off to give prochymosin including a pro-fragment. Prochymosin is a substantially inactive form of the enzyme which, however, becomes activated under acidic conditions to the active chymosin by autocatalytic removal of the pro-fragment. This activation occurs in vivo in the gastric lumen under appropriate pH conditions or in vitro under acidic conditions.
The structural and functional characteristics of bovine, ie Bos taurus, pre-prochymosin, prochymosin and chymosin have been studied extensively. The pre-part of the bovine pre-prochymosin molecule comprises 16 aa residues and the pro-part of the corresponding prochymosin has a length of 42 aa residues. The active bovine chymosin comprising 323 aa is a mixture of two forms, A and B, both of which are active, and sequencing data indicate that the only difference between those two forms is an aspartate residue at position 290 in chymosin A and a glycine residue at that position in chymosin B.
However, it is evident that the productivity in terms of overall yield of gene product is an important factor for the cost effectiveness of industrial production of the enzyme. Improvement of the productivity of chymosin would lead to a lower production costs and reduced use of resources. Accordingly, a continued industrial need exists to improve the productivity of chymosin in recombinant expression systems.
Additionally, there is a need for a chymosin with modified proteolytic activity under cheese-making conditions. In most cheeses, chymosin is responsible for “primary” proteolysis, which leads to the release of peptides that are later used by lactic acid bacteria for “secondary” proteolysis and flavour formation during ripening.